One of NCI's top priorities is to develop biomarkers that can preoperatively determine which among the computed tomography (CT)-found pulmonary nodules (PNs) are lung cancer. Tremendous efforts have been made to develop biomarkers by detecting cell-free (e.g., plasma) miRNAs directly released from primary lung tumors. Yet none of the biomarkers has been accepted in the clinical settings. Peripheral blood mononuclear cells (PBMCs) act as the first line of defense against malignancy in immune system, their dysfunction may occur as an early event in cancer immunogenicity or immune evasion. We have shown that the assessment of PBMC miRNAs may provide a new and immunological approach for the early detection of lung cancer. The objective of this application is to develop PBMC- based miRNA biomarkers for precisely identifying lung cancer among CT-found PNs in heavy smokers. There are two specific aims: 1), to systemically and comprehensively define PBMC miRNAs of lung cancer patients by using whole-transcriptome next-generation sequencing, and 2), to develop a panel of PBMC miRNA biomarkers for precisely identifying malignant PNs using droplet digital PCR. The success of this proposed R21-exploratory project will lead to a large and prospective study to comprehensively validate the biomarkers for lung cancer diagnosis. Future use of the biomarkers for discriminating malignant from benign PNs could spur early treatment of lung cancer, while minimizing complications from diagnostic and therapeutic procedures in smokers with benign diseases, and thus reduce lung cancer-associated deaths.